Microbial pathogens have developed a variety of strategies to infect the human host and cause disease. Many Gram-negative bacteria use type IV secretion systems (T4SSs) to deliver bacterial proteins, called effectors, into host cells. The effectors help to modulate signaling events within the host in order to create conditions favorable for bacterial survival. We are committed to the in-depth analysis of microbial virulence strategies. We use as a model organism the bacterium Legionella pneumophila, the causative agent of a potentially fatal respiratory infection known as Legionnaires' disease. Each year more individuals in the U.S. contract Legionnaires' disease (8,000 to 18,000) than there are cases of ALS (Amyotrophic Lateral Sclerosis or Lou Gehrig's Disease), thus making L. pneumophila a significant health threat and a considerable economic burden. Moreover, the infection cycle of L. pneumophila shows numerous parallels to the virulence programs of Salmonella, Chlamydia, Mycobacterium, Coxiella, and many other human pathogens that manipulate host cells from within a membrane-enclosed compartment. In addition, given that a type IV secretion system (T4SS), the major virulence apparatus of L. pneumophila, is present in numerous animal and plant pathogens including Helicobacter or Agrobacterium, the in-depth analysis of this translocation system and its cargo proteins, called effectors, is of great importance for our general understanding of microbial virulence. Last but not least, the effector proteins that are used by L. pneumophila to manipulate host cell processes display remarkable parallels to eukaryotic proteins, and deciphering their function will yield valuable insight into mechanistic and regulatory concepts about processes that occur within our own cells. Thus, obtaining a detailed understanding of Legionella's biology and its virulence strategies is essential to more effectively diagnose, treat, and prevent this dangerous pneumonia, and will profoundly improve people's lives and w L. pneumophila is ubiquitously found in freshwater habitats such as cooling towers, air conditioning systems, or water fountains. Major outbreaks of Legionnaires' disease occur when water from contaminated sources is aerosolized and subsequently inhaled by humans. That was the case during an outbreak of Legionnaires disease in New York City in 2015, where more than 120 individuals got infected and 12 died of the disease. Immune-compromised individuals, infants, or the elderly are at an elevated risk of contracting an infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires' disease cases within the U.S. has doubled over the past decade, making this microorganism is an emerging public health threat. Upon inhalation, L. pneumophila infects and replicates within alveolar macrophages, specialized immune cells within our lung. L. pneumophila delivers close to 300 proteins, called effectors, through a T4SS into the host cell. Most L. pneumophila effector proteins have not been characterized in detail, and their activities and host targets remain unknown. Interference with T4SS activity renders L. pneumophila avirulent, underscoring the important role of the translocated effectors for infection. Over the past funding period, we have made important progress in developing and applying new research tools to decipher the biological role of effectors. We revealed that during infection L. pneumophila translocates several effectors that mimic host cell proteins with E3 ubiquitin ligase activity. E3 ubiquitin ligases catalyze the final step in an enzymatic cascade that results in the transfer of the small protein ubiquitin from E2 ubiquitin-conjugating enzymes to a particular target protein. Poly-ubiquitination of target proteins alters their cellular fate, often resulting in their proteasomal degradation. By encoding its own E3 ligases, L. pneumophila can hijack the host cell ubiquitination machinery and use it for its own benefit. We found that one of the L. pneumophila effectors, in addition to exploiting host cell ubiquitination, takes advantage of yet another host cell machinery that controls S-palmitoylation, a reversible form of lipidation. The covalent attachment of a palmitoyl moiety to the L. pneumophila effector allows the protein to stably associate with the Golgi compartment, a cell organelle involved in protein secretion. Without S-palmitoylation, the L. pneumophila effector fails to properly localize to the Golgi, highlighting the importance of host cell-mediated S-palmitoylation for the function of this bacterial effector. Our studies suggest that pharmacological inhibition of S-palmitoylation may be a way to interfere with the localization and function of microbial virulence factors and to treat infections with L. pneumophila and related pathogens. In addition to the contributions described above, we also developed together with Drs. Joshua LaBaer and Ji Qiu (Arizona State University) an experimental pipeline for the comprehensive identification and validation of novel host-pathogen interactions between L. pneumophila effectors and human proteins. We employed a high throughput screening platform called NAPPA, a nucleic acid-programmable protein array composed of almost 13,000 human proteins, to detect stable interaction events between L. pneumophila effectors and their respective human targets. In addition, we adapted this platform to also monitor the modification of human proteins with adenosine monophosphate (AMP), a post-translational modification catalyzed by a variety of microbial virulence factors. By combining NAPPA with a set of in vitro and cell-based validation experiments, this pipeline has proven effective in detecting and characterizing L. pneumophila-human interactions. The flexibility of this technology also allows it to be adapted to the study of a large variety of microbial pathogens and their interactions with human host proteins. Together, these studies hold the key to obtaining an in-depth understanding not only of the virulence mechanisms of L. pneumophila and related pathogens but also of regulatory networks that exist within our own cells and that have been hijacked by L. pneumophila into its virulence program.